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BACKGROUND: Despite the recognized advantages of Peer-Assisted Learning (PAL) in academic settings, there is a notable absence of research analyzing its effects on students' Academic Burnout. This study aims to cover this gap by assessing the underlying effectiveness of Informal Peer-Assisted Learning (IPAL) as a cooperative learning method, focusing on its potential to mitigate academic burnout among medical students. METHODS: In 2022, a cross-sectional study was conducted at the School of Medicine, Universidad Central del Caribe, in Puerto Rico. The research team gathered data from 151 participants, 49.19% of 307 total student body. This cohort included 76 female students, 71 male students, and 4 individuals saying other. The School Burnout Inventory questionnaire (SBI-9) was employed to assess Academic Burnout, along with an added query about self-reported IPAL. The SBI-9 underwent validation processes to ascertain its reliability and validity, incorporating the Exploratory Factor Analysis and Confirmatory Factor Analysis. Following this, the investigators conducted an analysis to determine the correlation between academic burnout levels and involvement in IPAL. RESULTS: The validation process of the questionnaire affirmed its alignment with an eight-item inventory, encapsulating two principal factors that elucidate academic burnout. The first factor pertains to exhaustion, while the second encompasses the combined subscales of cynicism and inadequacy. The questionnaire shows high reliability (Cronbach's alpha = 0.829) and good fit indices (Comparative Fit Index = 0.934; Tucker-Lewis Index = 0.902; Standardized Root Mean Squared Residual = 0.0495; Root Mean Squared Error of Approximation = 0.09791; p-value < 0.001). The factors proven in the selected model were used to evaluate the correlation between Academic Burnout and IPAL. Students engaged in IPAL showed significantly lower academic burnout prevalence compared to those who never participated in such practices, with a mean academic burnout score of 44.75% (SD 18.50) for IPAL engaged students versus 54.89% (SD 23.71) for those who never engaged in such practices (p-value < 0.013). Furthermore, within the group engaged in IPAL, students displayed lower levels of cynicism/inadequacy 41.98% (SD 23.41) compared to exhaustion 52.25% (SD 22.42) with a p-value < 0.001. CONCLUSIONS: The results of this study underscore a notable issue of academic burnout among medical students within the surveyed cohort. The investigation reveals a significant correlation between Academic Burnout and IPAL, suggesting that incorporating IPAL strategies may be beneficial in addressing burnout in medical education settings. However, further research is needed to explore potential causal mechanisms.
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Grupo Paritario , Estudiantes de Medicina , Humanos , Estudiantes de Medicina/psicología , Femenino , Masculino , Estudios Transversales , Agotamiento Profesional , Encuestas y Cuestionarios , Puerto Rico , Adulto Joven , Adulto , Reproducibilidad de los Resultados , Educación de Pregrado en MedicinaRESUMEN
A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least across neuronal synaptic boutons. Released Arc particles can be accumulated in astrocytes after seizures. It is still not known how capsid assembling and transmission timescale is affected by seizures. This scientific field is relatively novel and is experiencing swift transformation as it grapples with difficult concepts in light of evolving experimental findings. We summarize the emergent literature on the subject and also discuss the specific rodent models for studying Arc effects in epilepsy. We summarized both to clarify the possible role of Arc-related pseudo-viral particles in epileptic disorders, which may be helpful to researchers interested in this growing area of investigation.
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Eye shine in the dark has attracted many researchers to the field of eye optics, but the initial studies of subwavelength arrangements in tapetum began only with the development of electronic microscopy at the end of the 20th century. As a result of a number of studies, it was shown that the reflective properties of the tapetum are due to their specialized cellular subwavelength microstructure (photonic crystals). These properties, together with the mutual orientation of the crystals, lead to a significant increase in reflection, which, in turn, enhances the sensitivity of the eye. In addition, research confirmed that optical mechanisms of reflection in the tapetum are very similar even for widely separated species. Due to progress in the field of nano-optics, researchers now have a better understanding of the main principles of this phenomenon. In this review, we summarize electron microscopic and functional studies of tapetal structures in the main vertebrate classes. This allows data on the microstructure of the tapetum to be used to improve our understanding of the visual system.
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Coroides , Vertebrados , Animales , Coroides/ultraestructura , Microscopía ElectrónicaRESUMEN
As do many other immunity-related blood cells, platelets release antimicrobial peptides that kill bacteria, fungi, and even certain viruses. Here we review the literature suggesting that there is a similarity between the antimicrobials released by other blood cells and the amyloid-related Aß peptide released by platelets. Analyzing the literature, we also propose that platelet-generated Aß amyloidosis may be more common than currently recognized. This systemic Aß from a platelet source may participate in various forms of amyloidosis in pathologies ranging from brain cancer, glaucoma, skin Aß accumulation, and preeclampsia to Alzheimer's disease and late-stage Parkinson's disease. We also discuss the advantages and disadvantages of specific animal models for studying platelet-related Aß. This field is undergoing rapid change, as it evaluates competing ideas in the light of new experimental observations. We summarized both in order to clarify the role of platelet-generated Aß peptides in amyloidosis-related health disorders, which may be helpful to researchers interested in this growing area of investigation.
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Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/metabolismo , Amiloidosis/inmunología , Plaquetas/inmunología , Encéfalo/inmunología , Enfermedad de Parkinson/inmunología , Animales , Autoanticuerpos/metabolismo , Modelos Animales de Enfermedad , HumanosRESUMEN
Amyloid beta (Aß) peptides have been implicated in both Alzheimer's disease (AD) and glaucoma and have been shown to be the key etiological factor in these dangerous health complications. On the other hand, it is well known that Aß peptide can be generated from its precursor protein and massively released from the blood to nearby tissue upon the activation of platelets due to their involvement in innate immunity and inflammation processes. Here we review evidence about the development of AD and glaucoma neuronal damage showing their dependence on platelet count and activation. The correlation between the effect on platelet count and the effectiveness of anti-AD and anti-glaucoma therapies suggest that platelets may be an important player in these diseases.
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Enfermedad de Alzheimer/sangre , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/metabolismo , Glaucoma/sangre , Enfermedad de Alzheimer/etiología , Glaucoma/etiología , HumanosRESUMEN
The amyloid beta (Aß) peptide and its deposits in the brain are known to be implicated in the neurodegeneration that occurs during Alzheimer's disease (AD). Recently, alternative theories views concerning both the source of this peptide and its functions have been developed. It has been shown that, as in all other known types of amyloidosis, the production of Aß originates in blood cells or cells related to blood plasma, from which it can then spread from the blood to inside the brain, with the greatest concentration around brain blood vessels. In this review, we summarize research progress in this new area and outline some future perspectives. While it is still unclear whether the main source of Aß deposits in AD is the blood, the possibility of blocking the chain of reactions that lead to constant Aß release from the blood to the brain may be exploited in an attempt to reduce the amyloid burden in AD. Solving the problem of Aß accumulation in this way may provide an alternative strategy for developing anti-AD drugs.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , HumanosRESUMEN
The ventral tegmental area (VTA) plays an important role in reward and motivational processes involved in drug addiction. Previous studies have shown that alpha1-adrenoreceptors (α1-AR) are primarily found pre-synaptically at this area. We hypothesized that GABA released onto VTA-dopamine (DA) cells is modulated by pre-synaptic α1-AR. Recordings were obtained from putative VTA-DA cells of male Sprague-Dawley rats (28-50 days postnatal) using whole-cell voltage clamp technique. Phenylephrine (10 µM; α1-AR agonist) decreased the amplitude of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of afferent fibers (n = 7; p < 0.05). Prazosin (1 µM, α1-AR antagonist), blocked this effect. Paired-pulse ratios were increased by phenylephrine application (n = 13; p < 0.05) indicating a presynaptic site of action. Spontaneous IPSCs frequency but not amplitude, were decreased in the presence of phenylephrine (n = 7; p < 0.05). However, frequency or amplitude of miniature IPSCs were not changed (n = 9; p > 0.05). Phenylephrine in low Ca(2+) (1 mM) medium decreased IPSC amplitude (n = 7; p < 0.05). Chelerythrine (a protein kinase C inhibitor) blocked the α1-AR action on IPSC amplitude (n = 6; p < 0.05). Phenylephrine failed to decrease IPSCs amplitude in the presence of paxilline, a BK channel blocker (n = 7; p < 0.05). Taken together, these results demonstrate that α1-ARs at presynaptic terminals can modulate GABA release onto VTA-DA cells. Drug-induced changes in α1-AR could contribute to the modifications occurring in the VTA during the addiction process.
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Neuronas Dopaminérgicas/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Área Tegmental Ventral/fisiología , Ácido gamma-Aminobutírico/metabolismo , Adrenérgicos/farmacología , Animales , Benzofenantridinas/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Estimulación Eléctrica , Indoles/farmacología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Masculino , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Técnicas de Placa-Clamp , Fenilefrina/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Prazosina/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas Sprague-Dawley , Técnicas de Cultivo de Tejidos , Área Tegmental Ventral/efectos de los fármacosRESUMEN
The Na(+)-dependent dopamine transporter (DAT) is primarily responsible for regulating free dopamine (DA) concentrations in the brain by participating in the majority of DA uptake; however, other DA transporters may also participate, especially if cocaine or other drugs of abuse compromise DAT. Recently, such cocaine-insensitive low-affinity mono- and poly-amine OCT transporters were described in astrocytes which use DA as a substrate. These transporters are from a different transporter family and while insensitive to cocaine, they are specifically blocked by quinine and some steroids. Quinine is inexpensive and is often found in injected street drugs as an "adulterant". The present study was designed to determine the participation of OCTs in cocaine dependent behavioral and physiological changes in mice. Using FVB mice we showed, that daily single injections of quinine (10 mg/kg, i.p.) co-administered with cocaine (15 mg/kg, i.p.) for 10 days significantly enhanced cocaine-induced locomotor behavioral sensitization. Quinine had no significant effect on the time course of behavioral activation. In astrocytes from the ventral tegmental area of mice, transporter currents of quinine-sensitive monoamine transporters were also augmented after two weeks of cocaine administration. The importance of low-affinity high-capacity transporters for DA clearance is discussed, explaining the known ability of systemically administered DAT inhibitors to anomalously increase DA clearance.
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Conducta Animal/efectos de los fármacos , Cocaína/farmacología , Quinina/farmacología , Animales , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Sinergismo Farmacológico , Locomoción/efectos de los fármacos , Masculino , RatonesRESUMEN
Thyroid hormones--particularly triiodothyronine, T3--play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre- or pro-metamorphic stages. Exposure to T3 in the water induced a rapid (beginning within 5 min) and significant decrease (approximately 20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI<2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI> or =2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.
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Locomoción/efectos de los fármacos , Locomoción/fisiología , Metamorfosis Biológica/efectos de los fármacos , Rana catesbeiana/crecimiento & desarrollo , Natación/fisiología , Triyodotironina/farmacología , Animales , Mejilla/fisiología , Fenómenos ElectrofisiológicosRESUMEN
INTRODUCTION: Few studies exist on thyroid status and ovarian dysfunction, although the prevalence of thyroid disease, particularly hypothyroidism, increases with advanced age and is more common in women. Loss of ovarian function is a lengthy process, and it is well known that follicle-stimulating hormone (FSH) increases with age and is correlated with loss of ovarian reserves. Limited information is available on FSH and thyroid-stimulating hormone (TSH) status in healthy euthyroid women in pre- and postmenopause states. We propose that patterns in FSH levels depend on the menopause state and that a possible relationship with TSH is present in mature women. METHODS: Baseline data from the study Health and Menopause in Hispanic Women in Puerto Rico were used. Eligible women were 30-84 years old. Demographic data and lifestyle and health information were collected through a questionnaire, and blood chemistries were analyzed. RESULTS: In women without thyroid disease, the median TSH was 1.97 mIU/L, and for euthyroid women the median was 1.84 mIU/L; no difference was observed between pre- and postmenopause states. A positive tendency was found between FSH levels and age in this group. CONCLUSIONS: This report compares the value of TSH in Puerto Rican women during pre- and postmenopausal states, and our findings are different from those in other ethnic groups. FSH levels correlate with age, and the general tendency of FSH to increase with age differs according to menopause state. No correlation between TSH and FSH levels was found in this study.
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Hormona Folículo Estimulante/sangre , Menopausia/sangre , Tirotropina/sangre , Adulto , Anciano , Anciano de 80 o más Años , Hispánicos o Latinos , Humanos , Persona de Mediana Edad , Puerto Rico/etnología , Análisis de Regresión , Encuestas y CuestionariosRESUMEN
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
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Acetilcolina/metabolismo , Mutación/genética , Síndromes Miasténicos Congénitos/genética , Unión Neuromuscular/genética , Receptores Nicotínicos/genética , Transmisión Sináptica/genética , Anciano , Sustitución de Aminoácidos , Animales , Señalización del Calcio/genética , Permeabilidad de la Membrana Celular/genética , Potenciales Postsinápticos Excitadores/genética , Femenino , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Cinética , Masculino , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Síndromes Miasténicos Congénitos/metabolismo , Síndromes Miasténicos Congénitos/fisiopatología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiopatología , Oocitos , Linaje , Xenopus laevisRESUMEN
A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues alphaS287, alphaC412, betaY441, gammaM299, gammaS460, deltaM293, deltaS297 and deltaN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [(125)I]-alpha-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC(50)), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC(50). Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.
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Lípidos de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Bungarotoxinas/farmacología , Expresión Génica , Humanos , Proteínas Musculares/genética , Oocitos/citología , Oocitos/metabolismo , Estructura Terciaria de Proteína/genética , Receptores Nicotínicos/genética , Proteínas Recombinantes de Fusión/genética , Xenopus laevisRESUMEN
We used tryptophan substitutions to characterize the beta M3 transmembrane domain (betaTM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the betaTM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [125I]-alpha-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the betaTM3 domain was consistent with an alpha-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed alpha-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica beta subunit.
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Mutagénesis Sitio-Dirigida , Subunidades de Proteína/química , Subunidades de Proteína/genética , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Triptófano/genética , Acetilcolina/farmacología , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Relación Dosis-Respuesta a Droga , Oocitos/metabolismo , Técnicas de Placa-Clamp , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/biosíntesis , Receptores Colinérgicos/biosíntesis , Torpedo , Xenopus laevisRESUMEN
The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the gammaM4 transmembrane domain (gammaTM4) by introducing tryptophan, at residues 451-462, along the gammaTM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [(125)I]alpha-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC(50), and Hill coefficient values for WT are 1.8 +/- 0.4 fmol, 30.3 +/- 1.1 microM, and 1.8 +/- 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 +/- 0.5, 3.6 +/- 0.6, and 3.0 +/- 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC(50) (19.5 +/- 1.7, 11.4 +/- 0.7, 16.4 +/- 3.8, and 19.1 +/- 2.6 microM, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC(50) (42.8 +/- 6.8 microM) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC(50) and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the gammaTM4.
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Sustitución de Aminoácidos , Receptores Colinérgicos/química , Torpedo , Triptófano , Animales , Microinyecciones , Oocitos , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Receptores Colinérgicos/genética , XenopusRESUMEN
Desensitization induced by chronic nicotine exposure has been hypothesized to trigger the up-regulation of the alpha4beta2 neuronal nicotinic acetylcholine receptor (nAChR) in the central nervous system. We studied the effect of acute and chronic nicotine exposure on the desensitization and up-regulation of different alpha4beta2 subunit ratios (1alpha:4beta, 2alpha:3beta, and 4alpha:1beta) expressed in Xenopus oocytes. The presence of alpha4 subunit in the oocyte plasmatic membrane increased linearly with the amount of alpha4 mRNA injected. nAChR function and expression were assessed during acute and after chronic nicotine exposure using a two-electrode voltage clamp and whole-mount immunofluorescence assay along with confocal imaging for the detection of the alpha4 subunit. The 2alpha4:3beta2 subunit ratio displayed the highest ACh sensitivity. Nicotine dose-response curves for the 1alpha4:4beta2 and 2alpha4:3beta2 subunit ratios displayed a biphasic behavior at concentrations ranging from 0.1 to 300 microm. A biphasic curve for 4alpha4:1beta2 was obtained at nicotine concentrations higher than 300 microm. The 1alpha4:4beta2 subunit ratio exhibited the lowest ACh- and nicotine-induced macroscopic current, whereas 4alpha4:1beta2 presented the largest currents at all agonist concentrations tested. Desensitization by acute nicotine exposure was more evident as the ratio of beta2:alpha4 subunits increased. All three alpha4beta2 subunit ratios displayed a reduced state of activation after chronic nicotine exposure. Chronic nicotine-induced up-regulation was obvious only for the 2alpha4: 3beta2 subunit ratio. Our data suggest that the subunit ratio of alpha4beta2 determines the functional state of activation, desensitization, and up-regulation of this neuronal nAChR. We propose that independent structural sites regulate alpha4beta2 receptor activation and desensitization.
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Neuronas/efectos de los fármacos , Nicotina/farmacología , Receptores Nicotínicos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Membrana Celular/metabolismo , Neuronas/metabolismo , XenopusRESUMEN
A recent tryptophan scanning of the alpha-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the alpha-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (alphaM282, alphaF284, alphaV285, alphaA287, and alphaI290) homologous to the alpha-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations alphaM282W and alphaV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., alphaM282 and alphaV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.
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Sustitución de Aminoácidos , Activación del Canal Iónico , Unión Neuromuscular/química , Subunidades de Proteína/química , Receptores Nicotínicos/química , Torpedo , Triptófano , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Bungarotoxinas/química , Regulación de la Expresión Génica/genética , Humanos , Activación del Canal Iónico/genética , Ratones , Datos de Secuencia Molecular , Unión Neuromuscular/genética , Oocitos/química , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Ratas , Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/genética , Homología de Secuencia de Aminoácido , Torpedo/fisiología , Triptófano/genética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The functional role of the alphaM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within alphaM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [(125)I]-alpha-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipid-exposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the alphaM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC(50). Taken together, our results suggest that alphaM3 contributes to the gating machinery of the nicotinic ACh receptor and that alphaM3 is comprised of a mixture of two types of helical structures.
Asunto(s)
Receptores Colinérgicos/fisiología , Secuencia de Aminoácidos , Animales , Bungarotoxinas/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Técnicas de Placa-Clamp , Conformación Proteica , Ensayo de Unión Radioligante , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Homología de Secuencia de Aminoácido , Torpedo , XenopusRESUMEN
Thyroid hormones (THs), primarily 3,3',5-triiode-(L)-thyronine (T(3)), have been clearly established as natural inducers of apoptosis during metamorphosis of anuran embryos. We decided to use this phenomenon to test the hypothesis that, prior to genomic activation, T(3) has acute actions in the neuromuscular junction (NMJ) of the tail of amphibian embryos. We detected a dramatic increase in the production of miniature end-plate currents (MEPCs) 2-5 min after continuous application of T(3) (250 nM) using focal recordings under voltage clamp. Furthermore, this increase in the spontaneous release of neurotransmitter, evaluated by the MEPC frequency, was maintained for several hours. Reverse-T(3), the "inhibitory" form of THs, prevented this increase in MEPC frequency, suggesting that this is probably a highly specific action of T(3). In addition, the elevation in MEPC frequency induced by T(3) was unchanged in the presence or absence of extracellular calcium. The T(3)-mediated increase in MEPC frequency was blocked by niflumic acid, a nonsteroidal antinflammatory fenamate used to prevent the apoptotic volume decrease observed in many systems. The present study demonstrated that T(3) induces a remarkable nongenomic action in the NMJ of the tadpole tail at pre- and promatamorphic stages.
Asunto(s)
Metamorfosis Biológica/fisiología , Unión Neuromuscular/efectos de los fármacos , Cola (estructura animal)/inervación , Triyodotironina Inversa/farmacología , Triyodotironina/farmacología , Acetilcolina/metabolismo , Animales , Calcio/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Espacio Extracelular/metabolismo , Técnicas In Vitro , Larva , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Neurotransmisores/metabolismo , Ácido Niflúmico/farmacología , Técnicas de Placa-Clamp , Rana catesbeiana , Cola (estructura animal)/efectos de los fármacos , Cola (estructura animal)/fisiología , Triyodotironina/antagonistas & inhibidoresRESUMEN
The retinae and brains of larval and adult amphibians survive long-lasting anoxia; this finding suggests the presence of functional K(ATP) channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the K(ATP) channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a: NeuroReport 12:1437-1441; Eaton et al., in press: NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (K(ATP)) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of K(ATP) channels (i.e., ATP- and spermine-free cytoplasm-dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch-clamp technique in whole-cell recordings, tolbutamide, a blocker of K(ATP) channels, blocks nearly 100% of the transient and about 30% of the steady-state inward currents and depolarizes the cell membrane by 5-12 mV. Second, inside-out membrane patches display a single-channel inward current induced by gluconate (40 mM) and blocked by ATP (200 microM) at the cytoplasmic side. The channels apparently show two sublevels (each of approximately 27-32 pS) with a total of 85-pS maximal conductance at -80 mV; the open probability follows a two-exponential mechanism. Thus, functional K(ATP) channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole-cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. K(ATP) channels may help maintain glial cell functions during ATP deficiency.
Asunto(s)
Membrana Celular/metabolismo , Larva/metabolismo , Neuroglía/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Ranidae/metabolismo , Retina/metabolismo , Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/deficiencia , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipoglucemiantes/farmacología , Inmunohistoquímica , Larva/citología , Larva/crecimiento & desarrollo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuroglía/citología , Neuroglía/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Rana catesbeiana , Rana pipiens , Ranidae/anatomía & histología , Ranidae/crecimiento & desarrollo , Receptores de Droga , Retina/citología , Retina/crecimiento & desarrollo , Receptores de Sulfonilureas , Tolbutamida/farmacología , Vasodilatadores/farmacologíaRESUMEN
We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome presenting in infancy in a single individual as progressive weakness and impaired neuromuscular transmission without overt degeneration of the motor endplate. Prolonged low-amplitude synaptic currents in biopsied anconeus muscle at 9 years of age suggested a kinetic disorder of the muscle acetylcholine receptor. Ultrastructural studies at 16 months, at 9 years, and at 15 years of age showed none of the typical degenerative changes of the endplate associated with the slow-channel congenital myasthenic syndrome, and acetylcholine receptor numbers were not significantly reduced. We identified a novel C-to-T substitution in exon 8 of the delta-subunit that results in a serine to phenylalanine mutation in the region encoding the second transmembrane domain that lines the ion channel. Using Xenopus oocyte in vitro expression studies we confirmed that the deltaS268F mutation, as with other slow-channel congenital myasthenic syndrome mutations, causes delayed closure of acetylcholine receptor ion channels. In addition, unlike other mutations in slow-channel congenital myasthenic syndrome, this mutation also causes delayed opening of the channel, a finding that readily explains the marked congenital weakness in the absence of endplate degeneration. Finally, we used serial morphometric analysis of electron micrographs to explore the basis for the progressive weakness and decline of amplitude of endplate currents over a period of 14 years. We demonstrated a progressive widening and accumulation of debris in the synaptic cleft, resulting in loss of efficacy of released neurotransmitter and reduced safety factor. These studies demonstrate the role of previously unrecognized mechanisms of impairment of synaptic transmission caused by a novel mutation and show the importance of serial in vitro studies to elucidate novel disease mechanisms.